Apparatus and method for measuring activity signals of biological samples

ABSTRACT

The present invention provides an apparatus for measuring activity signals of a biological sample comprising: a measurement chamber (A) storing a target liquid containing a biological sample; a porous insulating substrate ( 5 ) provided with a measurement electrode ( 1 ) on at least one side; and a conveying device ( 8 ) which conveys the target liquid stored in the measurement chamber (A) and passes the target liquid through the porous insulating substrate ( 5 ) from the measurement electrode ( 1 ) side, wherein the conveying device ( 8 ) is operated to trap the biological sample contained in the target liquid onto the measurement electrode ( 1 ), so that the activity signals of the biological sample are measured through the measurement electrode ( 1 ). According to the apparatus, activity signals emitted from the biological sample can be detected easily, rapidly and accurately.

TECHNICAL FIELD

The present invention relates to an apparatus and method for measuringactivity signals emitted from a biological sample such as a cell or thelike.

BACKGROUND OF THE INVENTION

Conventionally, physicochemical signals emitted in accordance with theactivities of biological samples are taken into a measurement apparatusand measured as electrical signals, or as digital signals, such as thefluorescence intensity signals that are emitted from the biologicalsample when an indicator is incorporated therein. For example, whenmeasuring the channel activation of a cell at the single channel level,an electrophysiology measurement apparatus provided with amicroelectrode probe, such as a patch clamp, and an exclusive controldevice is used to obtain digital signals showing the quantity ofelectricity passing through the channel. The obtained digital signalsare used to calculate the opening/closing periods, timing and frequencyof the channel for measuring channel activation.

The patch clamp method employs a micro portion (patch) of a cellmembrane attached to a tip of a micropipette so that ion transport via asingle channel protein molecule can be electrically recorded with themicroelectrode probe. Among cell biology techniques, the patch clampmethod is one of the few methods that permit the real-time examinationof the functions of a single protein molecule (e.g., “Molecular Biologyof the Cell”, 3rd edition, published by Garland Publishing Inc., NewYork, 1994, Japanese translation by Keiko NAKAMURA et al., pages 181 to182, published in 1995 by Kyoiku Sha).

A fluorescent dye method is used for measuring the electrical activityof a cell in combination of an image processor and a luminous indicatoror a fluorochrome that emits light depending on the concentrationvariations in a specified ion. For example, the ion mobility within acell is monitored based on the fluorescence images of a cell taken by aCCD camera. According to the fluorescent dye method, the ion channelactivation of an entire cell is generally determined by measuring thequantity of ions flowing into the cell by the fluorescence measurementmethod.

More specifically, the patch clamp method requires specializedtechniques for preparing and handling a micropipette and takes a lot oftime to measure one sample. Therefore, the patch clamp method is notsuitable for the high-speed screening of various types of drug candidatecompounds. In contrast, the fluorescent dye method permits thehigh-speed screening of various types of drug candidate compounds.However, the fluorescent dye method requires the step of dyeing thecell, which raises the problems of the high background caused by thedyestuff and the decolorization of the dyed cell with the passage oftime, resulting in a lowered S/N ratio at measurement.

Methods for observing electrical and chemical variations in biologicalsamples are disclosed in the prior art references of U.S. Pat. Nos.2,949,845, 5,810,725, 5,563,067 and 5,187,069, Japanese UnexaminedPatent Publication No. 1997-827318, and International Publication Nos.WO 01/25769, WO 98/54294, WO 99/66329 and WO 99/31503.

U.S. Pat. Nos. 2,949,845, 5,810,725 and 5,563,067 and JapaneseUnexamined Patent Publication No. 1997-827318 disclose an integratedcomplex electrode and a measurement system using the same which arecharacterized by forming microelectrodes on a glass substrate using aphotolithographic technique so as to extracellularly monitor theelectrical changes of a cell with a multipoint.

International Publication No. WO 01/25769 discloses forming perforationson an insulating substrate, and arranging biological samples such ascells or the like containing ion channels at the perforations, so that agiga-seal is formed on the cell or the like and the insulating substratesurface. The thus configured substrate uses reference electrodes andmeasurement electrodes that are disposed at two areas separated by thegiga-seal to measure the current that is generated when the ions passthrough the ion channels.

U.S. Pat. No. 5,187,069 discloses a device which culture cells on anelectrode and measures impedance changes to monitor cell proliferation.

International Publication No. WO 98/54294 discloses a device whichcauses a cell to adhere to a planar electrode for measuring electricalsignals.

International Publication No. WO 99/66329 discloses a device formonitoring the activities of a cell on a porous material according tovariations in resistance and impedance, and an assay method using thedevice.

International Publication No. WO. 99/31503 discloses a method using asubstrate provided with perforations wherein a patch clamp is formed bytrapping cells onto the perforations for use in measuring currentvariations.

Other prior arts of the present invention include Japanese UnexaminedPatent Publication Nos. 1999-326166, 1993-157728 and 1987-73152.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an apparatus and methodfor measuring activity signals of biological samples easily, rapidly andaccurately.

The above object of the present invention can be achieved by anapparatus for measuring activity signals of a biological samplecomprising: a measurement chamber storing a target liquid containing abiological sample; a porous insulating substrate provided with ameasurement electrode on at least one side; and a conveying device whichconveys the target liquid stored in the measurement chamber and passesthe target liquid through the porous insulating substrate from themeasurement electrode side. In the thus configured apparatus, theconveying device is operated to trap the biological sample contained inthe target liquid onto the measurement electrode, so that the activitysignals of the biological sample are measured through the measurementelectrode.

Moreover, the object of the present invention can be achieved by amethod for measuring activity signals of a biological sample comprisingthe steps of: injecting a target liquid containing a biological sampleinto a measurement chamber; trapping the biological sample onto ameasurement electrode by conveying the target liquid from themeasurement chamber and then passing the target liquid through a porousinsulating substrate provided with the measurement electrode on at leastone side; and measuring activity signals of the biological samplethrough the measurement electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the configuration of an essential partof an apparatus for measuring the activity signals of biological samplesaccording to Embodiment 1 of the present invention.

FIG. 2 is an enlarged view of the essential parts of the apparatus shownin FIG. 1.

FIGS. 3(A) and (B) schematically illustrate activities of biologicalsamples (cells).

FIG. 4 is a plan view schematically illustrating an apparatus formeasuring activity signals emitted from biological samples according toEmbodiment 2.

FIG. 5(A) is a plan view illustrating a cell separating unit of theapparatus shown in FIG. 4, and FIG. 5(B) is a cross sectional viewillustrating the same.

FIG. 6(A) is a plan view illustrating a porous insulating substrate ofthe apparatus shown in FIG. 4, and FIG. 6(B) is a cross sectional viewillustrating the same.

FIG. 7 is a cross-sectional view partially illustrating the apparatusshown in FIG. 4.

FIG. 8 is another cross-sectional view partially illustrating theapparatus shown in FIG. 4.

FIGS. 9(A) and (B) are block diagrams schematically illustrating anapparatus for measuring activity signals emitted from biological samplesaccording to Embodiment 3 of the present invention.

FIG. 10 shows the mean value variations of standard deviations for aCarbachol concentration.

FIGS. 11(A) and (B) are block diagrams schematically illustrating anapparatus for measuring activity signals emitted from biological samplesaccording to Embodiment 4 of the present invention.

FIG. 12 shows the results obtained by approximating the standarddeviation variations before and after the administration of Carbachol toa normal distribution.

FIG. 13 shows the results obtained by approximating the standarddeviation variations before and after the administration of Carbachol toa normal distribution according to a prior method.

FIG. 14 shows the distances obtained by comparing the mean value andhalf-widths of an obtained normal distribution, respectively, withreference values.

FIG. 15 is a block diagram schematically illustrating the configurationof an apparatus for measuring the activity signals of biological samplesaccording to another embodiment of the present invention.

FIG. 16 is a block diagram schematically illustrating the configurationof an apparatus for measuring the activity signals of biological samplesaccording to still another embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE PRESENT INVENTION

Hereinafter, the embodiments of the present invention will be describedwith reference to drawings.

EMBODIMENT 1

FIG. 1 schematically illustrates the configuration of an essential partof an apparatus for measuring the activity signals of biological samplesaccording to Embodiment 1 of the present invention. The apparatus has aconfiguration such that a measurement electrode 1 for detectingphysicochemical signals of biological samples such as a cell or the likeis formed on the upper surface (the surface on which the biologicalsample is placed) of a porous insulating substrate 5, and a conductor 2is derived from the measurement electrode 1. The physicochemical signalis a signal emitted from a biological sample such as a cell or the like.More specifically, it is a signal emitted from a specific portion of ameasurement target, such as the ion channel of a cell, or a signal whichvaries due to specific events occurring in the measurement target, suchas the activation of the ion channel or receptor of a cell in reactionto a drug.

A nylon mesh is employed as the porous insulating substrate 5 in thisembodiment. However, the porous insulating substrate 5 is not limitedthereto and cellulose mixed ester, hydrophilic polyvinylidenedifluoride, hydrophobic polyvinylidene difluoride,polytetrafluoroethylene, polycarbonate, polypropylene, polyethyleneterephthalate, etc., may be used. More specifically, Isopore (made ofpolyethylene terephthalate, product of Millipore) or Omnipore (made ofpolytetrafluoroethylene, product of Millipore) is preferably used.Generally, a porous insulating substrate 5 having a pore diameter of notless than 1 μm but not more than 1,000 μm and a thickness of not lessthan 1 μm but not more than 10,000 μm is selected. Typically, a porousinsulating substrate having a pore diameter of 5 μm and a thickness of10 μm can be used. However, a porous insulating substrate having athickness of 100 μm or more is preferably used.

The measurement apparatus is obtained as described below. A measurementelectrode 1 and a conductor 2 are initially formed on the porousinsulating substrate 5, preferably by sputtering a conductive material.According to this embodiment, gold is used as the conductive materialand plasma is generated by a high frequency between a pair of electrodesunder a low vacuum in the presence of an inert gas such as argon. Thegold is repelled from the cathode by ion energy, and forms on the porousinsulating substrate which is positioned on an anode opposite to thecathode. The electrode and the conductor can be also formed by a vacuumevaporation method or a printing method instead of the sputteringmethod. Platinum, copper, silver, a combination of silver and silverchloride, or a combination of platinum and platinum black can be usedinstead of gold as the conductive material. Moreover, a conductivematerial containing a conductive plastic can be used instead of metallicmaterials.

The measurement electrode 1 is preferably formed by sputtering without amask so that the electrode material is infused deeply into the porousinsulating substrate 5. In contrast, the conductor 2 is preferablyformed by pre-covering the porous insulating substrate 5 with a mask(not shown) for patterning so that the conductive material is preventedfrom infusing deeply into the porous insulating substrate 5.

In this embodiment, the measurement electrode 1 is formed in a diskshape, but it may take any shape depending on the measurement target.The measurement electrode 1 is not particularly limited in size, but inthis embodiment, it has approximately the same horizontal cross-sectionarea as a measurement chamber A, which will be described later.

As described above, the measurement electrode 1 and the conductor 2 areformed on the porous insulating substrate 5. Thereafter, the porousinsulating substrate 5 is clamped between a cell separating unit 3 and asupport 6. The cell separating unit 3 and the support 6 are eachprovided with an aperture, and the centers of the aperture of the cellseparating unit 3, the measurement electrode 1 formed on the porousinsulating substrate 5, and the aperture of the support 6 are disposedso as to be approximately in alignment. Thus, the aperture wall of thecell separating unit 3 and the porous insulating substrate 5 define themeasurement chamber A (corresponding to the entire space of the apertureformed in the cell separating unit 3 in FIG. 1). The cell separatingunit 3 and the support 6 are fixed to an adhesive layer 4 containingadhesive, which is provided around the aperture. The adhesive layer 4preferably has easy peelability and is watertight. For example,one-component RTV rubber (diacetate-type) KE42T (product of Shin-EtsuChemical Co., Ltd.) can be used.

A reference electrode 7 is disposed at a side wall of the measurementchamber A, and is immersed in a target liquid 23 which is stored in themeasurement chamber A. The reference electrode 7 provides a referenceelectric potential for detecting the activity signals of the biologicalsamples to be measured, and is composed of, for example, Ag—AgCl. Thetarget liquid 23 includes a DMEM culture medium. The cells to becultured include animal-derived cells.

The measurement chamber A is covered with a lid 21, which prevents thetarget liquid 23 from evaporating. The lid 21 is not necessarilyrequired, depending on the type and the measurement conditions of thetarget liquid 23, and thus a configuration with no lid 21 is alsoacceptable.

Subsequently, a suction line attachment 8 is fixed to a side of an undersurface of the support 6 with an adhesive layer 9 sandwichedtherebetween. The suction line attachment 8 consists of a suction unit 8a tapering from the bottom to the top and a suction line 8 b forconnection to the suction unit 8 a. The suction unit 8 a is positionedto be approximately in alignment to the aperture of the support 6. Thus,a suction chamber 8 is delimited by the porous insulating substrate 5,the aperture wall of the support 6 and the inner wall of the suctionunit 8 a. The suction line attachment 8 functions as a conveying devicefor sucking and conveying the target liquid 23, and is connected to asuction pump (not shown). The apparatus for measuring the activitysignals of biological samples can be thus configured.

Hereinafter, a method for measuring the activity signals of biologicalsamples using the above-mentioned apparatus will be described.Initially, the target liquid 23, such as a cell medium or the like, isinjected into the measurement chamber A. The amount of the target liquid23 is, for example, 50 μl.

When there is no suction force acting on the suction unit 8 a, theamount of the target liquid 23 that drops through the porous insulatingsubstrate 5 to the suction chamber B is very small. Generally, theamount is one-fiftieth or less of the total target liquid 23 stored inthe measurement chamber A.

Subsequently, the suction pump (not shown) is used to suck the targetliquid 23 stored in the measurement chamber A. When the suction chamberB is depressurized, the target liquid 23 passes through the porousinsulating substrate 5, and subsequently flows through the suction line8 b via the suction chamber B in the direction shown by the arrow. Incontrast, biological samples 25 contained in the target liquid 23 cannotpass through the porous insulating substrate 5, and are thus adsorbed tothe measurement electrode 1 as shown in FIG. 2. Activity signals emittedfrom the biological samples can be detected as an electrical potentialdifference generated between the measurement electrode 1 and thereference electrode 7. For example, when the biological sample 25 is acell in a static state as shown in FIG. 3(A), the opening and closing ofthe ion channels are in a state of equilibrium and conductance variesonly slightly between channels so the change in generated voltage issmall, potential amplitude in the vicinity of a cell membrane isapproximately uniform. In contrast, as shown in FIG. 3(B), when the cellis in an active state, the opening and closing of the ion channels arenot in a state of equilibrium, and conductance varies greatly betweenchannels so the change in generated voltage is large, and the potentialamplitude in the vicinity of the cell membrane is not uniform.Therefore, cell activity can be determined on the basis of time-seriesvariations in the detected electrical potentials.

After measuring, the inside of the apparatus is washed by feeding acleaning solution, such as a physical saline, into the measurementchamber A while operating the suction pump (not shown). The porousinsulating substrate 5 may be replaced, if necessary, before a differenttarget liquid 23 is injected into the measurement chamber A. Thus, theactivity signals of biological samples can be successively measured onvarious solutions containing drug candidate compounds.

As described above, according to the measurement method of the presentinvention, the target liquid 23 is simply supplied to the measurementchamber A and is sucked via the suction line attachment 8. Thus, thebiological samples 25 can be adsorbed to the measurement electrode 1, toincrease contact resistance, resulting in improved signal detectionsensitivity. Moreover, the suction line attachment 8 expedites theprocesses of renewing the target liquid 23 and cleaning. Consequently,the activity signals of biological samples can be measured easily,rapidly and accurately.

EMBODIMENT 2

FIG. 4 is a plan view schematically illustrating an apparatus formeasuring the activity signals of biological samples according toEmbodiment 2 of the present invention. The apparatus according toEmbodiment 2 is obtained by arranging in a matrix 16 of the measurementelectrodes 1 employed in the apparatus of Embodiment 1 shown in FIG. 1.In the present embodiment, the diameter of each measurement electrode 1is set to be 2 mm, and the space between adjacent respective measurementelectrodes 1 is set to be 1 mm. The measurement electrodes 1 are notparticularly limited to the number, arrangement, size, shape and thelike of this embodiment, but the measurement electrode 1 is preferred tohave, for example, an area of not less than 1 μm² but not more than 1cm², an approximately circular or rectangular shape, has a space of notless than 10 but not more than 10,000 μm between adjacent measurementelectrodes 1, to be arranged in a matrix. In this embodiment, the sameparts are designated by the same numerals as in Embodiment 1, and thusthe detailed descriptions are omitted.

In FIG. 4, the measurement electrodes 1 and the conductors 2 connectedthereto are arranged on the front side of the porous insulatingsubstrate 5. The porous configuration may be destroyed by using the heator laser beams between a plurality of conductors 2 on the front side ofthe porous insulating substrate 5 to thereby further increase theinsulation. The reference electrodes 7 and the conductors 12 connectedthereto are individually arranged on the inner wall and the top surfaceof the aperture of the cell separating unit 3 to correspond to eachmeasurement electrode 1. The cell separating unit 3 is usually composedof a transparent material.

FIG. 5(A) is a plan view and FIG. 5(B) is a cross-sectional view withreference to the cell separating unit 3. FIG. 6(A) is a plan view andFIG. 6(B) is a cross-sectional view with reference to the porousinsulating substrate 5. As shown in FIG. 5, a measurement chamber A isformed at each aperture of the cell separating units 3, in which aplurality of measurement electrodes (not shown) are disposed. As shownin FIG. 6(B), the measurement electrodes 1 are formed by sputteringconductive materials without a mask so that the material is infused intothe inside of the porous insulating substrate 5. Alternatively, theconductor 2 is formed by sputtering the conductive material through amask layer 10 formed on the top side of the porous insulating substrate5 so that the material is prevented from infusing into the inside of theporous insulating substrate 5. FIGS. 7 and 8 are cross sectional viewspartially illustrating the apparatus shown in FIG. 4. FIG. 7 is anenlarged view of area C. FIG. 8 is an enlarged view of area D. Thebiological samples 25 of a cell or the like contained in the targetliquid 23 are sucked by the suction line attachment (not shown) andadsorbed to the measurement electrode 1 in the same manner as inEmbodiment 1. The suction line attachment A is provided with a pluralityof suction units (corresponding to reference numeral 8 a in FIG. 1)corresponding to the respective measurement electrodes 1, and can causethe biological samples 25 to simultaneously adsorb to the respectivemeasurement electrodes 1 via the common suction line (corresponding toreference numeral 8 b in FIG. 1). Thus, the activity signals of thebiological samples can be measured under various conditions in a shorttime. The timing for the adsorption of the biological samples 25 to therespective measurement electrodes can be varied by providing anindividual suction line to each measurement electrode 1 instead of thecommon suction line.

EMBODIMENT 3

FIG. 9 is a block diagram schematically illustrating a configuration ofthe apparatus for measuring the activity signals of biological samplesaccording to Embodiment 3. The apparatus employs the apparatus ofEmbodiment 1 as a measurement unit (a signal source) 101, and has afunction for processing electrical signals detected by the measurementunit 101. As shown in FIG. 9(A), the apparatus is provided with a groupSD (standard deviation) calculation unit 102, a mean value calculationunit 105, an activation evaluation unit 120 and a data display unit 110.According to the group SD calculation unit 102, the standard deviationof one group consisting of a predetermined number of samples iscalculated on the basis of time-series data detected by the measurementunit 101. Groups consisting of the predetermined number of samples maybe temporally consecutive or may be spaced at predetermined timeintervals. According to the mean value calculation unit 105, the meanvalue of the obtained plurality of standard deviations is calculated.The activation evaluation unit 120 evaluates the activation of thebiological samples on the basis of the mean value of the standarddeviations. The activation evaluation unit 120 is provided with anactivation calculation unit 108 and an activation classification unit109. Therefore, according to the activation calculation unit 120,activation can be calculated based on the input information and can beclassified by comparison with pre-stored information. The data displayunit 110 displays the obtained activation. According to the apparatus,noise can be removed from digital signals (predetermined time-seriesdata) captured at a fixed sampling rate, and then significant signalsshowing, for example, the opening and closing of ion channels can beextracted, measured and classified.

As shown in FIG. 9(B), the group SD calculation unit 102, the mean valuecalculation unit 105 and the activation evaluation unit 108 can beconfigured into a computer containing a hard disk on which programs forthese calculations are recorded. The data display unit 110 can be a CRTdisplay. As shown in FIG. 9(B), the computer is further provided with anormal distribution approximation unit 103, a stimulation unit 104 and amean value/half-width calculation unit 106, which will be describedlater.

The apparatus of this embodiment configured as described above was usedto measure the effect of Carbachol, a chemical substance, on nerve cellsprepared from lymnaea. Carbachol is known as an analog to theacethylcoline neurotransmitter. Carbachol (a product of Sigma ChemicalCo.) was dissolved into an artificial brain-spinal fluid, and wassubsequently caused to act on nerve cells at concentrations of 0, 0.1,0.3, 1, 3, 10, 30 and 100 SM. Electrical signals emitted from theactivated cells were then measured. For each Carbachol concentration,time-series data were sampled every 100 ms from 10-second time-seriesdata obtained from the measurement unit 101, and standard deviations ofthe obtained data were calculated. FIG. 10 plots the mean values of theobtained standard deviations.

The mean value of the standard deviations represents variations in theelectrical potential in the vicinity of the cell membrane, and is usedfor evaluating the activation of ion channels. As can be seen from FIG.10, the mean value of the standard deviations increases with an increasein the Carbachol concentration, reaching its peak at the concentrationof 10 μM, and then decreases. As described above, the measurement methodand apparatus according to this embodiment verify that the activation ofion channels in the nerve cells of lymnaea depends on the Carbacholconcentration. Moreover, the activation of all channels of the nervecell can be inferred from the experiment results.

EMBODIMENT 4

FIG. 11 is a block diagram schematically illustrating a configuration ofan apparatus for measuring the activity signals of biological samplesaccording to Embodiment 4. As shown in FIG. 11(B), the apparatus isconfigured similarly to the apparatus of Embodiment 3 (see FIG. 9(B)).The mean value calculation unit 105 shown in FIG. 9(A) is not employed,whereas a normal distribution approximation unit 103 and a meanvalue/half-width calculation unit 106 are employed, resulting in aconfiguration as shown in FIG. 11(A).

The normal distribution approximation unit 103 classifies a plurality ofstandard deviations obtained in the group SD calculation unit 102 into aplurality of classes set at predetermined widths. The classes areplotted on the X axis and the number of standard deviations classifiedinto each class is plotted on the Y axis, to obtain a graph. The graphobtained is approximated to a normal distribution. Methods forapproximating the graph to a normal distribution include, for example,various curve fitting analyses, such as exponential decrease,exponential increase, Gaussian, Lorentzian, Sigma, Multipeak andnon-linear methods. The mean value/half-width calculation unit 106calculates the mean value and half-width (the half-peak height width) ofthe normal distribution obtained.

The apparatus of the present embodiment thus configured was used tomeasure how the chemical substance Carbachol acts on nerve cellsprepared from lymnaea. More specifically, the normal distributionapproximation unit 103 produced a frequency distribution of the standarddeviations on the basis of signals that were detected by the measurementunit 101 before and after Carbachol was administered to lymnaea nervecells at 50-μM concentration.

FIG. 12 shows a histogram approximated to a normal distribution, wherethe histogram was produced from standard deviations obtained throughcalculations made every 5 ms based on time-series data from 10-seconddetection signals before and after the Carbachol is administered. Thegraph on the left shows the state before administration, and the graphon the right shows the state after administration. As shown in FIG. 12,the administration of Carbachol increases the mean value and half-widthof the standard deviations. According to the calculation results of themean value/half-width calculation unit 106, the mean value is 0.478 andthe half-width is 0.109 before administration whereas the mean value is0.703 and the half-width is 0.175 after administration. The calculationresults show that the administration of Carbachol activates the ionchannels of the nerve cells of lymnaea, and thus the fluctuations of theactivity-related electrical potential caused by the opening and closingof the activated ion channels are expressed.

FIG. 13 shows the results obtained by comparing cell conditions beforeadministration and after administration by a prior intracellularrecording method under the same conditions as in the above experiment.In this embodiment, an extracellular recording method is employed as ameasurement method. A comparison of FIG. 12 with FIG. 13 indicates thatthe measurement results are the same between the intracellular recordingmethod and the extracellular recording method. As described above,according to the measurement method of the present invention, the cellactivities and variations thereof resulting from opening and closing ofthe ion channels can be measured easily without employing a priorintracellular recording method. Therefore, electrical variations in thebiological sample can be measured even without forming a high-resistanceseal (giga-seal) and the like between the biological sample and themeasurement device, which eliminates the possibility of damaging thebiological sample.

Furthermore, according to the present invention, the absolute values ofion channel activation and the increase or decrease of channelactivation can be compared before and after the administration of a drugto a cell or with respect to the administration amount. As describedlater, for example, drug effects can be classified qualitatively andquantitatively.

EMBODIMENT 5

The intracellular recording method verifies that activation of the Caion channels of smooth muscle cells is blocked depending on theconcentration of nifedipine when stimulated by a 10-μM concentration ofnorepinephline. In this embodiment, a method for evaluating drugs usingthe apparatus for measuring the activity signals of biological samplesaccording to the above Embodiment 4 will be described.

FIG. 14 shows a normal distribution of standard deviations produced fromdetection signals of the measurement unit 101, illustrating distancesobtained by comparing the mean values and half-widths of the normaldistribution of standard deviations, respectively, with referencevalues. The distances are represented as relative movement and relativespread. The relative movement and relative spread of nifedipine atvarious concentrations (0.1 μM to 100 μM) were stored as parameters in adatabase in advance and then the effects of two types of Ca channelblocking agents, A and B are classified.

As shown in FIG. 14, the relative movement and relative spread behaviorof compound A (Δ) are almost the same as those of nifedipine (◯) at eachconcentration, and compound A is thus estimated to be the same kind ofCa ion channel blocking agent as nifedipine. In contrast, the relativemovement and relative spread of compound B (□) scarcely vary even whenthe concentration varies, and compound B is remarkably different fromnifedipine (◯) in behavior. Therefore, compound (B) is a type of Ca ionchannel blocking agent that does not exist in smooth muscle cells. Inthis way, the effects of various drugs can be estimated.

Furthermore, drug screening can be effectively conducted by determiningwhether or not the distance from the reference value is within apredetermined value (e.g., within a range of ±5% from the referencevalue, as shown by the dashed-line circle) based on the relativemovement and relative spread shown in FIG. 14.

ANOTHER EMBODIMENT

The present invention has been described with reference to the aboveembodiments, but it is not limited thereto. In the above Embodiments 4and 5, for example, the distance obtained by comparing each of the meanvalue and the half-widths of the normal distribution obtained based ondetection signals with the reference value is used for evaluation, butparameters based on the standard deviation (or variance) of the obtainednormal distribution can also be suitably used to evaluate activity.

A configuration as shown in FIG. 15 can be obtained by further providingthe configuration described in Embodiments 4 and 5 and shown in FIG.11(A) with a sample dividing unit 111. The sample dividing unit 111 is avery effective means for analyzing the characteristics of various ionchannels existing on the cell membrane.

In the configuration as described in Embodiments 4 and 5 and shown inFIG. 11(A), only one measurement unit (signal source) 101 is provided,whereas when a plurality of measurement electrodes 101 are provided asdescribed in Embodiment 2, a configuration as shown in FIG. 16 can beobtained. In this measurement apparatus, a signal addition unit 107 addsthe activity signals generated in the selected single or pluralmeasurement units (signal sources) 101. When stimulation signals areprovided to the respective signal sources 101 from a stimulation unit104, biological samples can be simultaneously stimulated, which makes itpossible to synchronize the timing of a plurality of activity signals tobe added.

In each of the above embodiments, a suction line attachment is providedso as to suck the target liquid so that it passes through a porousinsulating substrate. As a means of conveying the target liquid,however, a pressurizing apparatus can be provided instead of the suctionline attachment so as to pressurize the measurement chamber, thuspassing the target liquid through the porous insulating substrate.

In the above Embodiment 3, the mean value calculation unit calculatesthe mean value based on a plurality of standard deviations that arecalculated by the group SD calculation unit 102. Alternatively, thisplurality of standard deviations can be divided into groups using aspecific constant in accordance with a time series and the mean valuecan be calculated per group. In the case where the mean value of eachgroup increases along the time series, a data display unit may beconfigured so as to display the time that the mean value exceeds apredetermined value and/or the time that the rate of increase of themean value falls below a predetermined value. This makes it possible toestimate the time lag until a chemical substance influences the cellactivity.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides an apparatus and amethod for measuring activity signals emitted from biological samples,thus enabling activity signals emitted from biological samples to bedetected easily, rapidly and accurately.

The present invention is applicable to high-speed medical screening orcell diagnosis (for example, discrimination between cancer cells andnormal cells), and can be carried out on-site during a surgicaloperation.

1-12. (canceled)
 13. An apparatus for measuring activity signals of abiological sample comprising: a measurement chamber storing a targetliquid containing a biological sample; a porous insulating substrateprovided with a measurement electrode on at least one side; and aconveying device which conveys the target liquid stored in themeasurement chamber and passes the target liquid through the porousinsulating substrate from the measurement electrode side, wherein theconveying device is operated to trap the biological samples contained inthe target liquid onto the measurement electrode, so that the activitysignals of the biological samples are measured through the measurementelectrode.
 14. An apparatus for measuring activity signals of abiological sample according to claim 13, further comprising a referenceelectrode that is provided in the measurement chamber.
 15. An apparatusfor measuring activity signals of a biological sample according to claim13, wherein the conveying device further comprises a suction apparatusfor sucking and then passing the target liquid through the porousinsulating substrate.
 16. An apparatus for measuring activity signals ofa biological sample according to claim 15, wherein the porous insulatingsubstrate is detachably provided between the measurement chamber and thesuction apparatus.
 17. An apparatus for measuring activity signals of abiological sample according to claim 13, wherein the porous insulatingsubstrate is composed of a resin film having a pore diameter of not morethan 1 μm but not less than 1,000 μm and a thickness of not more than 1μm but not less than 10,000 μm.
 18. An apparatus for measuring activitysignals of a biological sample according to claim 13, furthercomprising: a conductor electrically connected to the measurementelectrode, wherein the conductor is formed on one side of the porousinsulating substrate via a mask layer and the measurement electrode isinfused into the porous insulating substrate more deeply than theconductor.
 19. An apparatus for measuring activity signals of abiological sample according to claim 18, wherein the measurementelectrode and the conductor are formed by sputtering a conductivematerial onto a surface of the porous insulating substrate.
 20. Anapparatus for measuring activity signals of a biological sampleaccording to claim 13, wherein the measurement chamber and the conductorare provided in plural; the conveying device is provided with a commonsuction line communicating with each of the measurement chambers; andthe conveying device is operated to simultaneously trap the biologicalsamples contained in the target liquid stored in the measurementchambers onto the corresponding measurement electrodes.
 21. An apparatusfor measuring activity signals of a biological sample according to claim20, further comprising a plurality of reference electrodes provided inthe plurality of measurement chambers, respectively.
 22. An apparatusfor measuring activity signals of a biological sample according to claim13, further comprising: a means for calculating the standard deviationper a predetermined number of samples based on time-series data ofactivity signals outputted via the measurement electrode; a means forcalculating the mean value of a plurality of the obtained standarddeviations; a means for evaluating the activity of the biologicalsamples based on the obtained mean value of the standard deviations; anda means for displaying the results of the activity evaluation.
 23. Anapparatus for measuring activity signals of a biological sampleaccording to claim 13, further comprising: a means for calculating thestandard deviation per a predetermined number of samples based ontime-series data of activity signals outputted via the measurementelectrode; a means for classifying the obtained plurality of thestandard deviations into a plurality of classes set by a predeterminedwidth and approximating the classification results to the normaldistribution; a means for evaluating the activity of the biologicalsamples based on the obtained normal distribution; and a means fordisplaying the results of the activity evaluation.
 24. An apparatus formeasuring activity signals of a biological sample according to claim 23,further comprising: a means for calculating the mean value andhalf-width of the obtained normal distribution, wherein the activity isevaluated based on the obtained mean value and half-width of the normaldistribution.